1. Field of the Invention
The invention relates to methods for assessing the fertilization potential of mammalian spermatozoa and for inducing the acrosome reaction in such spermatozoa.
2. Description of the Prior Art
It has been known for some time that to be able to fertilize ovulated eggs ejaculated mammalian sperm must reside in the female reproductive tract for several hours; the precise time required varies from one mammal to another. The term "capacitation" was coined to describe this phenomenon. Similarly, sperm removed from the cauda epididymis for in vitro fertilization or for artificial insemination must be incubated under conditions that promote capacitation.
Capacitation has been defined as the process in the female (or in vitro) that prepares the spermatozoon to undergo the acrosome reaction and also quite probably to develop a whiplash or hyperactivated motility that may enhance ability to penetrate the zona pellucida (Yanagimachi, J. Reproduc. Fertil., 23:193-196 (1970)).
The acrosome is a membrane-bound organelle that appears during spermiogenesis as a product of the Golgi complex. It may be considered biochemically analogous to a lysosome. The size and morphology of the acrosome vary considerably from one mammal to another. However, it always occupies the interior region of the sperm head, just above the nucleus and beneath the plasma membrane. Acrosomal membranes underlying the plasma membrane and overlying the nuclear membrane are referred to as the outer and inner acrosomal membrane, respectively.
The acrosome reaction is an exocytotic event involving fusion of sperm plasma and outer acrosomal membranes at many sites, formation of hybrid membrane vesicles that are eventually sloughed from the sperm head, and exposure of acrosomal contents (including a variety of hydrolases, such as hyaluronidase, proteinases, glycosidases, lipases, and phosphatases) and inner acrosomal membrane. Among mammals, only sperm that have undergone the acrosome reaction can penetrate the zona pellucida and, upon reaching the perivitelline space, fuse with egg plasma membrane via plasma membrane overlying the postacrosomal region of the sperm head.
Among the many substances reported to induce mammalian sperm to undergo the acrosome reaction in vitro are serum albumin, .beta.-lactoglobulin, lysolecithin, ionophores, catecholamines, glycosaminoglycans, cyclic nucleotides, steroids, as well as components originating from blood, oviductal fluid, follicular fluid, cumulus cells, oocytes and the zona pellucida. This diversity has led to considerable confusion and controversy about both the nature of the acrosome reaction inducer and the site of the acrosome reaction in vivo. Since only sperm that have undergone the acrosome reaction are capable of penetrating the zona pellucida and fertilizing eggs, these are important issues.
A variety of techniques have been used to detect the acrosome reaction in vitro. Although light microscopy is sufficient to assess the status of the acrosome of some mammalian sperm, in many cases it has been necessary to employ transmission or scanning electron microscopy, or fluorescence microscopy to make an unambiguous assessment.
For an excellent review of the literature on sperm capacitation and the acrosome reaction, see Wassarman, Ann. Rev. Cell Biol, 3: 109-142 (1987).
Traditional methods for the evaluation of male infertility consist of taking a history, performing a physical examination and carrying out semen analysis. This last is the most widely used test for the evaluation of the fertility potential of male mammals, with special attention to sperm count, progressive motility and the morphology of the spermatozoa. The number of sperms able to undergo the acrosomal reaction is also ostensibly evaluated. However, on one hand, sperm count, motility and morphology give little information on the ability of the sperm to undergo the acrosomal reaction and to penetrate the egg; on the other, current methods to induce the acrosomal reaction, and therefore test sperm functional parameters, are time consuming and, at best, semiquantitative.
It has been recently suggested that in cases of unexplained infertility in a man judged fertile by standard semen analysis, the fertilizing capacity of spermatozoa could be tested by assaying total seminal acrosine, a trypsin-like enzyme contained in the acrosome which is released during the acrosome reaction and is involved in the fertilization process. The assay technique involves chemical disruption of the acrosome and measurement of the total acrosine content of the sperms. However, there is no firm evidence that the total acrosine content correlates with sperm fertilization ability. The only available test that gives reliable information on the sperm fertilization potential, i.e. the hamster egg penetration test, is not only expensive but also difficult for routine application.
Similarly, no simple, reliable and inexpensive techniques are available for increasing the ability of spermatazoa to undergo the acrosome reaction and, hence, increase the fertilization potential of the sperm. Improved techniques are actively being sought in order to more effectively deal with human and animal male fertility problems.